INTERMEDIATES IN SYNAPTIC VESICLE RECYCLING REVEALED BY OPTICAL IMAGING OF DROSOPHILA NEUROMUSCULAR-JUNCTIONS

We show that uptake and release of the styryl dye FM1-43 may be used t o monitor synaptic vesicle exocytosis and recycling at Drosophila larv al neuromuscular junctions. At Drosophila nerve terminals, FM1-43 spec ifically labels subsynaptic domains enriched in synaptotagmin, in a ma nner that requires Ca2+, membrane depolarization, and shibire (shi) fu nction. Endocytosis rates, very low in unstimulated synapses, are indu ced severalfold by the exocytosis of synaptic vesicles. Using shi(ts1) mutant synapses to separate synaptic vesicle fusion and recycling tem porally, we show that recycling events subsequent to the shi block do not require extracellular Ca2+. We suggest that two distinct intermedi ate stages in vesicle recycling may be trapped and analyzed at Drosoph ila neuromuscular junctions.